DEVELOPMENT OF RAPID IDENTIFICATION METHOD OF PATHOGENIC VIBRIOS

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Bacteria of genus Vibrio are normal habitant in natural aquatic environments, and include some pathogenic species such as Vibrio cholerae, V.parahaemolyticus. Because of existence of many vibrios species similar to the pathogenic species, many days are necessary for identification of the pathogenic strain isolated from natural environment. Furthermore, simple and rapid method is absolutely necessary for specimens from septicemia patients, because only immediate antibiotics treatment can save life of the patient. Immunological or genetical methods are excel in the specificity and able to make possible rapid identification without time-consuming biochemical tests. For example, polymerase chain reaction (PCR) does not need cultivation of the bacterium. The present study is performed to establish simple and rapid identification methods of V.vulnificus, a pathogenic vibrio causing fetal septicemia to patients having underlying disease such as hepatic dysfunction.Hemolysin of V.vulnificus was chosen as a tool of immunological and genetical methods. To obtain enough amount of immunogen, purification method of the hemolysin was modified and established. Specificity of the hemolysin in the species was confirmed with antiserum prepared with the purified hemolysin. Primers for PCR are synthesized and applied for ED-PCR which is a PCR combined with a enzyme-linked immunosorbent assay. Although some blood components interfered the PCR,a pretreatment (lysis of erythrocytes and washing) eliminated the interfere. Specificity of the PCR for V.vulnificus was demonstrated. Mechanism of hemolysin action and immunologocal detection method of siderophores, chelator for acquisition of iron, of V.vulnificus and V.parahaemolyticus were also studied.